This disclosure pertains to Multiscale Spectral Nanoscopy (MSN) and is generally related to the disclosure in U.S. Pat. No. 7,982,194—Single Nanoparticle Tracking Spectroscopic Microscope (SNTSM) which is incorporated herein in its entirety. The study of molecular interactions in biological systems has long been hampered by the inability to observe molecular phenomena on their native length scale (down to <1 nm) and on a time scale relevant to molecular dynamics (<millisecond), while simultaneously placing these interactions and dynamics in their macromolecular biological context. The traditional methods of observing molecular biology in context have been optical imaging methods, starting with the most predominantly used and commercially available method: confocal microscopy.
Traditional Imaging Methods:
Modern day laser scanning confocal microscopes have the ability to provide optical sections at up to 8 Hz. Unfortunately, these speeds are not sufficient for the evaluation of biological processes which happen in real time in three dimensions. Confocal microscopes have been further improved by the implementation of Nipkow spinning disks, which allow the acquisition of 3D volumes at up to 1 Hz, with near confocal performance. However, these time scales (>1 second) are still far too slow to monitor chemical dynamics at the cellular or subcellular level.
Fast Large Scale 3D Imaging Methods:
To solve the problem of imaging large volumes with high temporal resolution selective plane illumination microscopy (SPIM) use an excitation beam that is spread out by a cylindrical lens and delivered to the sample perpendicular to the collection objective, creating a plane of illumination which allows for optical sectioning. Fast imaging of volumes of 400×400×200 um of neuronal action potentials every 6 seconds have been achieved, although the axial resolution is on the order of 5 microns. This method has been improved by rapidly scanning a laser beam to create an illumination plane, allowing for more intense illumination and faster acquisition times, acquiring volumes of 1000×1000×600 um in 60-90 seconds with 300 nm lateral and 500 nm axial resolution.
Superresolution Methods:
While the above methods have opened doors to the study of larger dynamics in developmental biology, they are still limited by diffractive nature of propagating beams of light, with the ultimate resolution limit on the order of 200 nm in the lateral dimensions and 600 nm in the axial dimensions. To bypass this limit, so-called “super-resolution” methods have been developed. Stimulated emission depletion microscopy (STED) uses a high power laser pulse to effectively turn off fluorescence emission in a certain area. By carefully shaping the laser pulse to define a spherical area around the focal spot, the focal spot size can be effectively reduced in size due to the depletion of surrounding emission. Isotropic focal spots with resolution down to 30 nm have been used to image mitochondrial cristae in live cells. Unfortunately, this is still a point scanning technique and has limited time resolution at larger spatial scales.
Other methods have relied on localization of single photoswitchable fluorophores. These methods (stochastic optical reconstruction microscopy, STORM; photoactivated localization microscopy, PALM) were initially implemented for investigating phenomena in two dimensions, but have been extended to three dimensions for up to a few microns through the use of astigmatic imaging (STORM, 20-30 nm lateral, 60 nm axial), biplane imaging (BP-PALM, 30 nm lateral, 75 nm axial), interferometry (iPALM, 20 nm lateral, 10 nm axial), or implementing a double helix PSF (DH-PSF, 10 nm lateral, 10 nm axial). Unfortunately, all of these methods are limited by their axial extent (usually only 1-2 microns) or temporal resolution (PALM and STORM require the observation of many photoswitching events, requiring tens of seconds to acquire a single image).
Some methods have endeavored to combined fast 3D imaging with superresolution, such scanning multifocal multiphoton 4Pi-confocal microscopy (MMM-4Pi), in which a two photon excitation beam is broken into 16-64 beamlets which scan a small subsection of the optical slice. By stitching these subregions together and scanning axially through the specimen, volumes of 10×10×5 μm can be observed in as little as 150 seconds, with isotropic resolution from 100-140 nm. Another method which aims to combine the benefits of superresolution with larger scale observations is the structured illumination microscope (SIM). By implementing excitation fields with frequencies near the spatial frequencies of the sample, a lower beat frequency, observable by a traditional microscope objective, can be created. This method has shown the ability to improve the lateral resolution down to ˜110 nm. This method has also been extended to three dimensions (3D-SIM) with volume imaging rates of 0.20 Hz for volumes of 25×25×2.72 um.
Despite these faster superresolution methods, there is still a huge range of length and time scales that go unobserved. For instance, the single-molecule methods such as DH-PSF and iPALM show remarkable spatial resolution, but they cover a relatively short length scale due to their small axial extent. Very few methods adequately address timescales below 1 msec. It would be desirable to provide systems and methods that address these and other shortcomings of existing systems.